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Fluorescence, Phosphorescence, & Chemiluminescence

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Presentation on theme: "Fluorescence, Phosphorescence, & Chemiluminescence"— Presentation transcript:

1 Fluorescence, Phosphorescence, & Chemiluminescence
A) Introduction 1.) Theory of Fluorescence and Phosphorescence: 10-5 to 10-8 s fluorescence 10-4 to 10s phosphorescence 10-14 to s 10-8 – 10-9s M*  M + heat - Excitation of e- by absorbance of hn. - Re-emission of hn as e- goes to ground state. Use hn2 for qualitative and quantitative analysis

2 Fluorescence, Phosphorescence, & Chemiluminescence
A) Introduction 1.) Theory of Fluorescence and Phosphorescence: Method Mass detection limit (moles) Concentration detection limit (molar) Advantages UV-Vis 10-13 to 10-16 10-5 to 10-8 Universal fluorescence 10-15 to 10-17 10-7 to 10-9 Sensitive For UV/Vis need to observe Po and P difference, which limits detection For fluorescence, only observe amount of PL

3 Example of Phosphorescence
2.) Fluorescence – ground state to single state and back. Phosphorescence - ground state to triplet state and back. 10-5 to 10-8 s 10-4 to 10 s Spins paired No net magnetic field Spins unpaired net magnetic field Fluorescence Phosphorescence 0 sec 1 sec 640 sec Example of Phosphorescence

4 3) Jablonski Energy Diagram
S2, S1 = Singlet States T1 = Triplet State Numerous vibrational energy levels for each electronic state Resonance Radiation - reemission at same l usually reemission at higher l (lower energy) Forbidden transition: no direct excitation of triplet state because change in multiplicity –selection rules.

5 4.) Deactivation Processes:
a) vibrational relaxation: solvent collisions - vibrational relaxation is efficient and goes to lowest vibrational level of electronic state within 10-12s or less. - significantly shorter life-time then electronically excited state - fluorescence occurs from lowest vibrational level of electronic excited state, but can go to higher vibrational state of ground level. - dissociation: excitation to vibrational state with enough energy to break a bond - predissociation: relaxation to vibrational state with enough energy to break a bond

6 4.) Deactivation Processes:
b) internal conversion: not well understood - crossing of e- to lower electronic state. - efficient since many compounds don’t fluoresce - especially probable if vibrational levels of two electronic states overlap, can lead to predissociation or dissociation.

7 4.) Deactivation Processes:
c) external conversion: deactivation via collision with solvent (collisional quenching) - decrease collision  increase fluorescence or phosphorescence ‚ decrease temperature and/or increase viscosity ‚ decrease concentration of quenching (Q) agent. Quenching of Ru(II) Luminescence by O2

8 4.) Deactivation Processes:
d) intersystem crossing: spin of electron is reversed - change in multiplicity in molecule occurs (singlet to triplet) - enhanced if vibrational levels overlap - more common if molecule contains heavy atoms (I, Br) - more common in presence of paramagnetic species (O2)

9 kf + ki + kec+ kic + kpd + kd
5.) Quantum Yield (f): ratio of the number of molecules that luminesce to the total number of excited molecules. - determined by the relative rate constants (kx) of deactivation processes f = kf kf + ki + kec+ kic + kpd + kd f: fluorescence I: intersystem crossing ec: external conversion ic: internal conversion pd: predissociation d: dissociation Increase quantum yield by decreasing factors that promote other processes Fluorescence probes measuring quantity of protein in a cell

10 6.) Types of Transitions:
- seldom occurs from absorbance less than 250 nm ‚ 200 nm => 600 kJ/mol, breaks many bonds - fluorescence not seen with s*  s - typically p*  p or p*  n

11 ‚ fluorescence especially favored for rigid structures
7.) Fluorescence & Structure: - usually aromatic compounds ‚ low energy of p p* transition ‚ quantum yield increases with number of rings and degree of condensation. ‚ fluorescence especially favored for rigid structures < fluorescence increase for chelating agent bound to metal. Examples of fluorescent compounds: quinoline indole fluorene 8-hydroxyquinoline

12 resonance forms of aniline
8.) Temperature, Solvent & pH Effects: - decrease temperature  increase fluorescence - increase viscosity  increase fluorescence - fluorescence is pH dependent for compounds with acidic/basic substituents. ‚ more resonance forms stabilize excited state. Fluorescence pH Titration resonance forms of aniline

13 Change in fluorescence as a function of cellular oxygen
9.) Effect of Dissolved O2: - increase [O2]  decrease fluorescence ‚ oxidize compound ‚ paramagnetic property increase intersystem crossing (spin flipping) Change in fluorescence as a function of cellular oxygen Am J Physiol Cell Physiol 291: C781–C787, 2006.

14 B) Effect of Concentration on Fluorescence or Phosphorescence
power of fluorescence emission: (F) = K’Po(1 – 10 –ebc) K’ ~ f (quantum yield) Po: power of beam ebc: Beer’s law F depends on absorbance of light and incident intensity (Po) At low concentrations: F = 2.3K’ebcPo deviations at higher concentrations can be attributed to absorbance becoming a significant factor and by self-quenching or self-absorption. Fluorescence of crude oil

15 C) Fluorescence Spectra
Excitation Spectra (a) – measure fluorescence or phosphorescence at a fixed wavelength while varying the excitation wavelength. Emission Spectra (b) – measure fluorescence or phosphorescence over a range of wavelengths using a fixed excitation wavelength. Phosphorescence bands are usually found at longer (>l) then fluorescence because excited triple state is lower energy then excited singlet state.

16 D) Instrumentation - basic design ‚ components similar to UV/Vis
‚ spectrofluorometers: observe both excitation & emission spectra. - extra features for phosphorescence ‚ sample cell in cooled Dewar flask with liquid nitrogen ‚ delay between excitation and emission

17 Fluorometers A-1 filter fluorometer
- simple, rugged, low cost, compact - source beam split into reference and sample beam - reference beam attenuated ~ fluorescence intensity A-1 filter fluorometer

18 Spectrofluorometer Perkin-Elmer 204
- both excitation and emmision spectra - two grating monochromators - quantitative analysis Perkin-Elmer 204

19 E) Application of Fluorescence
- detect inorganic species by chelating ion Ion Reagent Absorption (nm) Fluorescence (nm) Sensitivity (mg/ml) Interference Al3+ Alizarin garnet R 470 500 0.007 Be, Co, Cr, Cu, F-,NO3-, Ni, PO4-3, Th, Zr F- Al complex of Alizarin garnet R (quenching) 0.001 Be, Co, Cr, Cu, F-,Fe, Ni,PO4-3, Th, Zr B4O72- Benzoin 370 450 0.04 Be, Sb Cd2+ 2-(0-Hydroxyphenyl)-benzoxazole 365 Blue 2 NH3 Li+ 8-Hydroxyquinoline 580 0.2 Mg Sn4+ Flavanol 400 0.1 F-, PO43-, Zr Zn2+ - green 10 B, Be, Sb, colored ions 8-Hydroxyquinoline flavanol alizarin garnet R benzoin

20 F) Chemiluminescence Examples:
- chemical reaction yields an electronically excited species that emits light as it returns to ground state. - relatively new, few examples A + B  C*  C + hn Examples: Chemical systems - Luminol (used to detect blood) - phenyl oxalate ester (glow sticks)

21 Luciferase gene cloned into plants
2) Biochemical systems - Luciferase (Firefly enzyme) “Glowing” Plants Luciferase gene cloned into plants Luciferin (firefly)

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